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Fig. 8. UAP56 does not mediate the ATP-dependent intra-nuclear mobility of SRm160. HeLa cells were co-transfected with mRFP-SRm160 and either EGFP-UAP56 wt or EGFP-UAP56 K95N. After 24 hours, the mRFP-SRm160 in a nuclear region of interest (white square) was photobleached for 3 seconds using maximum laser intensity at 568 nm. The recovery of mRFP-SRm160 in the bleached zone was recorded. The cells were later permeabilized with digitonin, which permeabilizes the cell membrane but leaves the nuclear envelope intact. When digitonin-permeabilized cells were photobleached, there was no recovery of fluorescence in the bleached zone (data not shown) (Wagner et al., 2004). Adding back 1 mM ATP restored fluorescence in cells expressing either wild-type UAP56 or UAP56 K95N. The ATP-dependence of SRm160 exchange at speckled domains did not depend on the ability of UAP56 to bind ATP. (A) A single cell for each experiment is shown before and after the photobleach, and then at one recovery time point. Scale bars: 10 µm. (B) The calculated recovery curves for all cells in each live-cell experiment are shown with the number of cells noted on each graph. (C) Matching calculated results for in vitro FRAP experiments in which the cells were digitonin-permeabilized before photobleaching. The error bars are standard deviations.
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